Extracellular nucleotides that activate G protein-coupled P2Y receptors (P2YRs) are attractive pharmaceutical targets due to their ability to modulate various functions in many tissues and organs under normal and pathophysiological conditions (Hillmann et al., 2009; Burnstock and Verkhratsky, 2009). Extracellular nucleotides and dinucleotides have been shown to play a role in ocular physiology and physiopathology (Crooke et al., 2008), and have been suggested as therapeutic agents for dry eye, retinal detachment and glaucoma (Guzman-Aranguez et al., 2007).
Ocular hypertension, the most common cause of glaucoma, is a target for agents that reduce intraocular pressure (IOP) (Pintor, 2005). When topically applied to New Zealand white rabbits, some nucleotides, e.g., diadenosine triphosphate and diadenosine pentaphosphate, produce an increase in IOP while others such as ATP, adenosine tetraphosphate and diadenosine tetraphosphate decrease IOP (Peral et al., 2009; Pintor et al., 2003, 2004).
Receptors for extracellular nucleotides including P2Y1, P2Y2 and P2Y4 have been identified in trabecular meshwork (TM) cells, an area of tissue in the eye that is responsible for draining the aqueous humor (Soto et al., 2005). Among these P2YR subtypes, activation of the P2Y1R by the selective agonist 2-MeS-ADP reduces aqueous humor outflow in bovine ocular. Other studies have reported the presence of P2Y1 and P2Y2 receptors in bovine TM cells, and of P2Y1, P2Y4 and P2Y11 receptors in a human TM cell line (Crosson et al., 2004).
Lately, the structure-activity relationship of P2Y6-R agonists and antagonists, and the molecular modeling of the P2Y6-R involved in the reduction of IOP have been extensively investigated (El-Tayeb et al., 2006; Jacobson et al., 2009; Costanzi et al., 2005; Maruoka et al., 2010; Besada et al., 2006); however, no potent and selective P2Y6-R agonist or antagonist has yet been identified. The development of agonists for the P2Y6-R included modification of the UDP phosphate chain, ribose ring, and base. Different uracil modifications have been performed over the last years in an attempt to identify agonists which will be more potent than the endogenous ligand UDP (Maruoka et al., 2010; Ginsburg-Shmuel et al., 2010; Ko et al., 2008).
The therapeutic potential of nucleotides in general, and for the treatment of glaucoma in particular, is limited, since they are degraded by extracellular enzymes, which reduce their potency, efficacy and duration of action. In addition, although nucleotides are chemically stable in a pH range of 4-11 (El-Tayeb et al., 2006), they are rapidly degraded at a more acidic or basic pH. Nucleotides are hydrolyzed enzymatically by the ecto-nucleoside triphosphate diphosphohydrolase family of ectonucleotidases, i.e., e-NTPDase and alkaline phosphatases (Nahum et al., 2002), and ecto-nucleotide pyrophosphatases/phosphodiesterases, i.e., e-NPPs (Grobben et al., 2000; Zimmermann, 2001). Therefore, there is a need for identification of both enzymatically and chemically stable nucleotide scaffolds that can be used to develop selective and potent P2YR agonists.
A few attempts to improve the stability of nucleotides have been reported (Cusack et al., 1987; Misiura et al., 2005; Kowalska et al., 2007), including the use of phosphate bioisosteres of nucleotides such as phosphonate (Eliahu et al., 2009; Joseph et al., 2004), phosphoramide (Zhou et al., 2005), and boranophosphate analogues (Nahum et al., 2002; Boyle et al., 2005; Barral et al., 2006; Eliahu et al., 2009).
A second strategy for enhancing stability of potential P2Y-R agonists is the use of dinucleotides, e.g., diuridine triphosphates, which show greater stability than analogues of mononucleotides (Shaver et al., 2005; Yerxa et al., 2002). Indeed, dinucleotides have been successfully developed before as P2Y2-R agonists. Thus, Up4U (INS365, Diquafosol) and Up4dC (INS37217, Denufosol) have been clinically tested for the treatment of dry eye disease and cystic fibrosis, respectively, however, both compounds did not show satisfying results at phase 3 clinical trial (http://www.businesswire.com/news/home/20110103005364/en; Jacobson and Boeynaems, 2010).
Ginsburg-Shmuel et al. (2010) discloses 5-OMe-UDP as a P2Y6-receptor agonist. As particularly shown, 5-OMe-UDP adopts the anti-conformation that is favored by the receptors, and the S sugar puckering that is the conformation preferred by the P2Y6-receptors but not the P2Y2- or P2Y4-receptors, and thus fulfills the conformational and H-bonding requirements of P2Y6-receptors and making a potent P2Y6-receptor agonist (EC50=0.08 μM vs. 0.14 μM for UDP).
U.S. Pat. No. 7,084,128 discloses a method of reducing IOP by administration of certain mono- or di-nucleotides, preferably mono- or diadenosine, mono-, di-, tri-, tetra-, penta- or hexaphosphate derivatives, or a pharmaceutically-acceptable salt thereof. The particular compounds exemplified are 2′-(O)-,3′-(O)-(benzyl) methylenedioxy-adenosine-5′-triphosphate and 2′-(O)-,3′-(O)-(benzyl)methylene dioxy-2″-(O)-,3″-(O)-benzyl methylene dioxy-P1,P4-di(adenosine 5′-)tetra phosphate, and as shown, at a concentration of 0.25 mM, these compounds produced a time dependent reduction in IOP, which was maximal at 1-2 hours with a reduction of 21-22%.
Eliahu et al. (2010) discloses certain non-hydrolyzable adenosine di- or triphosphate analogues such as 2MeS-adenosine-β,γ-CH2-5′-triphosphate and 2MeS-adenosine-β,γ-CCl2-5′-triphosphate as potent agents for reducing IOP. As stated in this publication, 2MeS-adenosine-β,γ-CCl2-5′-triphosphate reduced IOP in normotense rabbits by 32% (EC50=95.5 nM), wherein the duration of effect was about 5 hours, i.e., was found to be more effective at reducing IOP than several common glaucoma drugs and thus represents a promising alternative to timolol maleate, which cannot be used for the treatment of patients suffering from asthma or cardiac problems.